I. Adenovirus Vectors
Adenoviruses have attracted increasing attention as expression vectors, especially for human gene therapy (Berkner, Curr. Top. Microbiol. Immunol., 158:39-66 (1992)). This is because the virus particle is relatively stable, and can be prepared as a high titer stock (10.sup.9 plaque forming units/ml) without purification. In addition, adenoviruses are useful because they can infect non-replicating cells. Further, adenovirus vectors have been proven safe and effective in humans. However, the following limitations have prevented their general use:
(1) The expression of adenovirus proteins in infected cells is believed to trigger a cellular immune response that precludes long-term expression of the transferred gene (Stratford-Perricaudet et al, Hum. Gene Ther., 1:241 (1990); Ginsberg et al, Proc. Natl. Acad. Sci., USA, 88:1651 (1991); Yang et al, Proc. Natl. Acad. Sci., USA, 91:4407 (1994); Dai et al, Proc. Natl. Acad. Sci., USA, 92:1401 (1995); Jaffe et al, Nat. Genet., 1:372 (1992); Li et al, Hum. Gene Ther., 4:403 (1993); Engelhardt et al, Hum. Gene Ther., 4:759 (1993); Simon et al, Hum. Gene Ther., 4:771 (1993); and Smith et al, Nat. Genet., 5:397 (1993)); and PA1 (2) The insert capacity of currently available adenovirus vectors is limited to about 8.0 kb of foreign DNA (Bett et al, Proc. Natl. Acad. Sci., USA, 13:8802 (1994)). PA1 (i) a first adenovirus inverted terminal repeat, PA1 (ii) a foreign gene, and PA1 (iii) a second adenovirus inverted terminal repeat, PA1 (i) a first adenovirus inverted terminal repeat, PA1 (ii) an adenoviral VAI gene and/or VAII gene, PA1 (iii) a gene foreign to adenovirus, wherein said gene is operably linked to a promoter functional in adenovirus target cells, and PA1 (iv) a second adenovirus inverted terminal repeat,
Hence, broad application of in vivo gene transfer for the treatment of inherited or acquired diseases requires a substantial improvement of existing systems, or the development of new viral or non-viral vector systems.
A. Reduction of Immunogenicity
In order to reduce the expression of adenovirus proteins, and thus reduce immunogenicity, and in order to prevent viral replication, the current adenovirus vectors have deletions in the E1 and/or E3 regions of the adenovirus genome. All of the other essential viral proteins are encoded by the adenovirus vector itself. E1 proteins can be complemented by culturing the E1 adenoviruses in human 293 cells. The E3 region is dispensable for growth of the virus in vitro.
Recent efforts have been directed at the deletion of additional regions (E2, E4) of the adenovirus genome, which encode early viral functions, in an attempt to further reduce viral gene expression after transduction of the target cells with the adenovirus vector (Engelhardt et al, Proc. Natl. Acad. Sci., USA, 91:6196 (1994); Yang et al, Nature Genet., 7:362 (1994); Zhou et al, Gene Therapy and Molecular Medicine, Keystone Symposia on Molecular and Cellular Biology, Steamboat Springs, Colo., Mar. 26-Apr. 1, 1995; Perricaudet et al, Ibid; and Finer et al, Ibid). To propagate these adenovirus vectors, cell lines have been developed that can provide the deleted functions. However, theoretically, it is very difficult, if not impossible, to provide all of the deleted adenovirus functions by a complementing cell line without substantially compromising the high adenovirus titer, which is currently one of the major advantages of adenovirus vectors.
B. Increasing the Capacity of Adenoviruses to Carry Foreign Genes
The lower packaging limit of adenovirus is unknown. However, the upper packaging limit of Ad5 is approximately 38 kb (Bett et al, J. Virol., 67:5911-5921 (1993)). As a result, adenovirus vectors with deletions of both the E1 and E3 sequences, about 6.0 kb in total, have a capacity for insertion of foreign DNA of up to approximately 8.0 kb.
After repeated passaging of permissive cells infected at a high multiplicity of infection (hereinafter "m.o.i.") with different adenovirus serotypes, subgenomic DNAs preferentially containing the left end of the adenovirus genome are packaged into adenovirus particles, and can be partially separated from wild-type adenovirus particles by cesium chloride (CsCl) density gradient centrifugation (Hammarskjold et al, Cell, 20:787-795 (1980)). In addition, after repeated passaging of permissive human KB cells infected at a high m.o.i. with Ad12, hybrid viruses containing symmetrically duplicated chromosomal DNA of the KB cell line flanked by a 700-1150 bp DNA fragment from the left terminus of Ad12 are produced (Deuring et al, Proc. Natl. Acad. Sci., USA, 78:3142-3146 (1981); Doerfler, Curr. Top. Microbiol. Immunol., 101:127-193 (1982); and Deuring et al, Gene, 26:283-289 (1983)). These hybrid viruses can be partially separated from Ad12 by CsCl equilibrium density gradient, and also can be propagated over years together with Ad12. However, the purity of these particles appears to be very low.
SV40/Ad5 hybrid viruses containing a total of 35 kb which comprise 5.5 copies of the SV40 genome and only 3.5 kb DNA from the left end of Ad5 have also been reported (Gluzman et al, J. Virol., 45:91-103 (1983)). The smallest genome size among the different types of Ad5/SV40 hybrid viruses is about 25 kb (Hassell et al, J. Mol. Biol., 120:209-247 (1978)).
It has recently been determined that the sequences required in cis for replication and packaging of adenovirus DNA comprise less than 500 bp (Grable et al, J. Virol., 64:2047-2056, (1990); and Hearing et al, J. Virol., 61:2555-2558 (1987)).
All of the cis-elements for packaging and replication are contained in 380 bp from the left end of the genome and 103 bp from the right end of the genome (Sussenbach et al, In: Current Topics in Microbiology and Immunology, Vol. 109, Doerfler, Ed. Springer-Verlag, Berlin, pp. 53-73 (1983); Tamanoi et al, In: Current Topics in Microbiology and Immunology, Vol. 109, Doerfler, Ed. Springer-Verlag, Berlin, pp 75-87 (1983); Hearing et al, supra; and Grable et al, supra).
It was postulated in the present invention that an adenovirus vector could be prepared in which all of the regions of the adenovirus genome were deleted, except for the packaging signal, and the inverted terminal repeats, containing the replication signal. Thus, it was postulated in the present invention that it is possible to accommodate up to 37 kb of foreign DNA into defective adenovirus vectors by supplying all of the proteins in trans from a helper virus or cell line. As a result, it was postulated in the present invention that it would be possible to deliver multiple or large genes containing tissue-specific or inducible promoters, as well as marker genes, in one vector, in cis. Such a vector would not encode any viral proteins, and thus would not be toxic or immunogenic to the host. Hence, the above-discussed problem of the immune response of the host arising from expression of viral proteins from the known adenovirus vectors might also be diminished.
Helper-dependent adenovirus vectors encoding the SV40 T antigen have been previously reported (Mansour et al, Proc. Natl. Acad. Sci., USA, 82:1359-1363 (1985); and Yamada et al, Proc. Natl. Acad. Sci., USA, 82:3567-3571 (1985)). However, these vectors, which were used to overproduce the polyoma T antigens (Mansour et al, supra) and the HSV thymidine kinase gene (Yamada et al, supra), had to be selected for by their growth in monkey cells. The T antigen provides a helper function, which overcomes the block to adenovirus growth in simian cells. However, since the T antigen of the tumor virus, SV40, is able to transform cells to a cancerous state (Hunter, Sci. Amer., 251:70-79 (1994)), it cannot be used in any application in humans.
It was postulated in U.S. patent application Ser. No. 08/488,014, filed Jun. 7, 1995, which is incorporated by reference herein in its entirety, that the use of a selection step could be avoided by a gene transfer vector comprising, in 5' to 3' orientation, the following elements:
wherein one or both of element (i) and element (iii) additionally comprise an adenovirus packaging signal; and that CsCl centrifugation could be employed to purify recombinant adenovirus containing the same, wherein the recombinant adenovirus is obtained by co-transfection of said gene transfer vector and adenovirus genomic DNA (see also, Mitani et al, Proc. Natl. Acad., Sci., USA, 92:3854-3858 (1995); Kochanek et al, Proc. Natl. Acad., Sci., USA, 93:5731-5736 (1996); and Clemens et al, Gene Therapy, 3:965-972 (1996)).
However, with the above-discussed gene transfer vector, there is the disadvantage in that multiple serial passages are required to obtain sufficient quantities of recombinant adenovirus. This is because the percentage of recombinant adenovirus produced is still a small percentage of the total amount of adenovirus produced upon co-transfection of the gene transfer vector with the adenovirus genomic DNA.
The present invention has overcome the above-described problem, without increasing the immunogenicity of the recombinant virus due to the production of adenovirus proteins, by incorporating into the gene transfer vector an adenovirus VAI gene and/or VAII gene, and the use of adenovirus genomic DNA which has a defective VAI gene and/or VAII gene during co-transfection. The VA genes encode small RNAs of 150-170 nucleotides in length, synthesized by the host RNA polymerase III. Ad2 expresses two VA RNAs, a major species VAI and a minor species VAII--although not all adenoviruses are endowed with two such RNAs (Ma et al, J. Virol. 67: 6605 (1993)). In Ad2 infected cells, VAI and VAII RNAs are expressed in equal amounts early in the infection cycle, whereas in the late phase of infection, the transcription rate of VAI RNA is strongly induced. VA RNAs are very stable and accumulate to high levels, possibly because of their compact secondary structure (Mathews, In: Current Topics in Microbiology and Immunology, Vol. 199, Doerfler and Boehm, Eds. Springer-Verlag, Berlin, pp. 173-187 (1995); and Pruzin et al, In: Current Topics in Microbiology and Immunology, Vol. 199, Doerfler and Boehm, Eds. Springer-Verlag, Berlin, pp. 201-226 (1995)). VAI RNA plays an important role in late viral protein synthesis in adenovirus infected cells (Thimmappaya et al, Cell, 31:543 (1982)). In cells infected with adenovirus having a deletion of the VA genes, initiation of translation of late viral proteins is strongly reduced (Schneider et al, Cell, 37:291 (1984)). VAI RNA binds to interferon and double-stranded RNA inducible eIF-2.alpha. kinase, and thus prevents its activation. In its activated form, this kinase inhibits translation by phosphorylation of the translation initiation factor eIF-2.alpha. (Thimmappaya et al, supra; Babich et al, Mol. Cell. Biol., 3:1212 (1983); Schneider et al, supra; and Siekierka et al, Proc. Natl. Acad. Sci., USA, 82:1959 (1985)). Thus, adenovirus genomic DNA with defective VA genes alone has poor amplification capacities.